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Monitoring this process, particularly synaptic vesicle recycling, in living cells has been instrumental in revealing the mechanisms responsible for neurotransmitter release. However, currently available reporters suffer from limitations, such as large probe sizes or limited compatibility for human neurons, hampering the quantitative analysis of synaptic pathophysiology. Here, we describe the NbLumSyt1 toolkit, a panel of nanobody-based affinity probes that target the luminal domain of the synaptic vesicle protein Synaptotagmin 1 (Syt1). These new tools enable quantitative, noninvasive imaging and functional interrogation of Syt1 exo-endocytosis and trafficking in human neurons, with unprecedented precision, versatility and cost efficiency, in technologies ranging from fixed- and live-cell super-resolution imaging to electron microscopy and mass spectrometry. Overall, NbLumSyt1 nanobinders provide a valuable platform for studying synaptic physiology and pathophysiology, benefiting fundamental neuroscience and translational efforts to study and develop treatments for brain-related disorders.<\/jats:p>\n                  <jats:p>\n                    <jats:bold>Graphical Abstract<\/jats:bold>\n                  <\/jats:p>","DOI":"10.1186\/s12951-026-04489-w","type":"journal-article","created":{"date-parts":[[2026,5,14]],"date-time":"2026-05-14T10:54:36Z","timestamp":1778756076000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["A versatile nanobody platform for live and super-resolution imaging of synaptic vesicle dynamics and plasticity in rodent and human 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